The dynamin family of proteins consists of unique GTPases involved in membrane fission and fusion events throughout the cell. The founding member, dynamin, is crucial for endocytosis, synaptic membrane recycling, membrane trafficking within the cell and more recently, has been associated with filamentous actin. Dynamin was first implicated in endocytosis when it was discovered to be the mammalian homologue of the shibire gene product in Drosophila. A temperature sensitive shibire allele causes a defect in clathrin-mediated endocytosis. Since then, overexpressing human dynamin mutants in mammalian cells was found to block clathrin-mediated endocytosis. Over the years, our cryo-EM structural work has played a leading role in dissecting the function of dynamin in membrane fission. We have shown that purified dynamin readily assembles into rings and spirals and it forms similar structures on liposomes, generating dynamin-lipid tubes that constrict upon GTP hydrolysis. A potential mechanism for dynamin constriction was revealed when we solved the three dimensional structure of dynamin in the non-constricted and constricted states by cryo-electron microscopy (cryo-EM). These results suggest dynamin wraps around the necks of budding vesicles as a helical polymer and upon GTP hydrolysis undergoes a significant constriction that ultimately leads to membrane fission. Recently, we solved a 3.75 resolution cryo-EM structure of the membrane-associated helical polymer of human dynamin-1 in the GMPPCP-bound state. Images for the high-resolution map was collected at the New York Structural Biology Center (NYSBC) in New York City using a FEI Krios microscope with a K2 direct electron detector. Our new dynamin helical map allowed us to build an atomic model of the assembled dynamin polymer bound to lipid. Comparing soluble crystal structures to our new high-resolution cryo-EM structure revealed conformational changes that occur upon assembly and lipid binding. The structure defines the helical symmetry of the dynamin polymer and the positions of its oligomeric interfaces, which were validated by cell-based endocytosis assays in collaboration with Dr. Justin Taraska, NHLBI. In addition, we solved the structure of the dynamin polymer in a post-hydrolysis state (10 resolution) that resembled the transition-state-defective dynamin mutant (K44A) that was previously solved in the lab. These post-hydrolysis conformations are super-constricted with a 3.4 nm inner lumen and assemble in 2-start helical arrays. Constriction of the membrane to 3.4 nm lumen is reaching the theoretical limit required for spontaneous membrane fission, supporting the model that dynamin alone is capable of causing membrane fission. Computational docking indicates that the ground state conformation of the dynamin polymer is sufficient to achieve this super-constricted pre-fission state and reveals how a 2-start helical symmetry promotes the most efficient packing of dynamin tetramers around the membrane neck. We recently improved the resolution of the dynamin-K44A map to 4.5 and are working toward a 3-4 map to build a molecular model. In the past year, we collaborated with Dr. Elizabeth Chen (UT Southwestern) to discover the structural interactions between dynamin and actin during muscle development. Our cryo-tomograms of co-assembled actin and dynamin reveals a mechanism for dynamin mediated actin bundling. In previous years, we collaborated with Drs. Sandra Schmid (UT Southwestern) and Vadim Frolov (U Basque Country) to explore the effect of dynamins powerstroke defined by the large swing of the BSE in dynamin. To dissect the fission reaction into stages, we utilized intra-molecular chemical cross-linking to stabilize dynamin in a conformation mimicking its transition-state. We found that dynamin trapped in the transition state is unable to mediate full fission, but forms stable hemifission intermediates without phosphate release. Dynamin assembly and augmented membrane insertion of its pleckstrin homology domain drives the hemifission state. Our findings, which are consistent with molecular simulations of the fission reaction, reveal a second, unappreciated energy barrier for full fission. Thus additional conformational dynamics are required after hemifission that enable dynamin to utilize the energy of GTP hydrolysis to complete the fission reaction. Previously, we also collaborated with Drs. Sambuughin (Uniformed Services University), Goldfarb (NINDS, NIH), Renwick (Queens University, Kingston Canada), Platonov (Ammosov North-Eastern Federal University, Russian Federation) and Toro (NHGRI, NIH) to characterized a dynamin mutant that leads to a rare case of Hereditary Spastic Paraplegia (HSP). This was the first report linking a mutation in dynamin-2 to HSP. In addition, the mutation is located in a region of dynamin distinct from all other dynamin-2 disease causing mutations.